Methods of protein purification and recovery

Abstract

Improved methods for purification and recovery of interferon-beta (IFN-β) and compositions comprising substantially monomeric IFN-β are provided. In one purification method, substantially purified IFN-β or variant thereof is precipitated and then dissolved in a guanidine hydrochloride (HCl) solution. Renaturation of the protein occurs by dilution with a suitable buffer. A similar purification method absent the precipitation step is also provided. Following renaturation of the IFN-β, residual guanidine HCl is removed by diafiltration or dialysis with a pharmaceutically acceptable buffer to prepare pharmaceutical compositions comprising substantially monomeric IFN-β.

1. A pharmaceutical composition having a pH of about 3.0 to about 5.0 and comprising interferon-beta (IFN-β), wherein said composition is prepared by a method consisting of:
 a) denaturing IFN-β with guanidine hydrochloride (HCl); 
 b) renaturing the IFN-β via dilution into a first buffer to obtain a renatured IFN-β solution comprising residual guanidine HCl; and 
 c) removing said residual guanidine HCl from said renatured IFN-β solution by diafiltration or dialysis of said renatured IFN-β solution into a second buffer that is pharmaceutically acceptable, wherein said second buffer is selected from the group consisting of aspartic acid and sodium succinate. 
 
     
     
       2. The pharmaceutical composition of  claim 1 , wherein said first buffer has a pH of about 3.0 to about 5.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 1.6 M or less. 
     
     
       3. The pharmaceutical composition of  claim 2 , wherein said first buffer has a pH of about 3.0 to about 4.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 0.2 M or less. 
     
     
       4. The pharmaceutical composition of  claim 3 , wherein said first buffer has a pH of about 3.0, and wherein said residual guanidine HCl is present in said renatured IFN-β solution at a concentration of 0.1 M or less. 
     
     
       5. The pharmaceutical composition of  claim 1 , wherein said composition comprises substantially monomeric IFN-β. 
     
     
       6. The pharmaceutical composition of  claim 1 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. 
     
     
       7. The pharmaceutical composition of  claim 1 , wherein said IFN-β is glycosylated or unglycosylated. 
     
     
       8. The pharmaceutical composition of  claim 1 , wherein said IFN-β is recombinantly produced. 
     
     
       9. The pharmaceutical composition of  claim 1 , wherein said IFN-β has at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and wherein said IFN-β retains the ability to bind to IFN-β receptors. 
     
     
       10. The pharmaceutical composition of  claim 1 , wherein said composition is injectable. 
     
     
       11. The composition of  claim 1 , wherein said first buffer is selected from the group consisting of aspartic acid and sodium succinate. 
     
     
       12. A pharmaceutical composition having a pH of about 3.0 to about 5.0 and comprising interferon-beta (IFN-β ), wherein said composition is prepared by a method consisting of:
 a) obtaining a sample comprising substantially purified IFN-β; 
 b) mixing said sample with guanidine hydrochloride (HCl) to obtain a first solution comprising solubilized denatured IFN-β; 
 c) diluting said first solution into a first buffer to obtain a second solution comprising solubilized renatured IFN-beta and residual guanidine HCl; and 
 d) removing residual guanidine HCl from said second solution by diafiltration or dialysis of said second solution into a second buffer that is pharmaceutically acceptable, wherein said second buffer is selected from the group consisting of aspartic acid and sodium succinate. 
 
     
     
       13. The pharmaceutical composition of  claim 12 , wherein said composition comprises substantially monomeric IFN-β. 
     
     
       14. The pharmaceutical composition of  claim 12 , wherein said first buffer has a pH of about 3.0 to about 5.0, and wherein said residual guanidine HCl is present in said second solution at a concentration of 1.6 M or less. 
     
     
       15. The pharmaceutical composition of  claim 14 , wherein said first buffer has a pH of about 3.0 to about 4.0, and wherein said residual guanidine HCl is present in said second solution at a concentration of 0.2 M or less. 
     
     
       16. The pharmaceutical composition of  claim 15 , wherein said first buffer has a pH of about 3.0, and wherein said residual guanidine HCl is present in said second solution at a concentration of 0.1 M or less. 
     
     
       17. The pharmaceutical composition of  claim 12 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. 
     
     
       18. The pharmaceutical composition of  claim 12 , wherein said IFN-β is glycosylated or unglycosylated. 
     
     
       19. The pharmaceutical composition of  claim 12 , wherein said IFN-β is recombinantly produced. 
     
     
       20. The pharmaceutical composition of  claim 12 , wherein said IFN-β has at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and wherein said IFN-β retains the ability to bind to IFN-β receptors. 
     
     
       21. The pharmaceutical composition of  claim 12 , wherein said composition is injectable. 
     
     
       22. The composition of  claim 12 , wherein said first buffer is selected from the group consisting of aspartic acid and sodium succinate. 
     
     
       23. A composition comprising substantially monomeric interferon-beta (IFN-β) and having a pH of about 3.0 to about 5.0, wherein said composition is prepared by a method consisting of:
 a) preparing a sample comprising substantially purified IFN-β; 
 b) mixing said sample with guanidine hydrochloride (HCl) to obtain a first solution comprising solubilized denatured IFN-β; and 
 c) renaturing said IFN-β by dilution of said first solution with a buffer, wherein said buffer has a pH of about 3.0 to about 5.0 and is selected from the group consisting of aspartic acid and sodium succinate. 
 
     
     
       24. A pharmaceutical composition comprising the composition of  claim 23 . 
     
     
       25. The composition of  claim 23 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. 
     
     
       26. The composition of  claim 23 , wherein said IFN-β is glycosylated or unglycosylated. 
     
     
       27. The composition of  claim 23 , wherein said IFN-β is recombinantly produced. 
     
     
       28. The composition of  claim 23 , wherein said IFN-β has at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and wherein said IFN-β retains the ability to bind to IFN-β receptors. 
     
     
       29. A pharmaceutical composition having a pH of about 3.0 to about 5.0 and comprising interferon-beta (IFN-β), wherein said composition is prepared by a method consisting of:
 a) denaturing IFN-β with guanidine hydrochloride (HCl); 
 b) renaturing the IFN-β via dilution into a first buffer to obtain a renatured IFN-β solution comprising residual guanidine HCl, wherein said first buffer has a pH of about 3.0 to about 5.0 and is selected from the group consisting of aspartic acid and sodium succinate; and 
 c) removing said residual guanidine HCl from said renatured IFN-β solution by diafiltration or dialysis of said renatured IFN-β solution into a second buffer that is pharmaceutically acceptable, wherein said second buffer is selected from the group consisting of aspartic acid and sodium succinate. 
 
     
     
       30. The pharmaceutical composition of  claim 29 , wherein said composition comprises substantially monomeric IFN-β. 
     
     
       31. The pharmaceutical composition of  claim 29 , wherein said second buffer is aspartic acid. 
     
     
       32. The pharmaceutical composition of  claim 29 , wherein said IFN-β has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. 
     
     
       33. The pharmaceutical composition of  claim 29 , wherein said IFN-β is glycosylated or unglycosylated. 
     
     
       34. The pharmaceutical composition of  claim 29 , wherein said IFN-β is recombinantly produced. 
     
     
       35. The pharmaceutical composition of  claim 29 , wherein said IFN-β has at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:1 as calculated using the ALIGN program with a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and wherein said IFN-β retains the ability to bind to IFN-β receptors.